This invention relates to a method for the production of antihemophilic factor (AHF or Factor VIII) and cold-insoluble globulin (CIg or fibronectin).
For a number of years, cryoprecipitation as described by Pool et al. has been a principal method for the isolation and partial purification of AHF. Nature, London 203, 312 (1964); New Eng. J. Med. 273, 1443-7 (1965). AHF can thus be prepared from single units of plasma in routine blood banking procedures such as in closed bag collection systems as illustrated, e.g., in U.S. Pat. Nos. 3,986,506 and 4,025,618, or concentrates can be prepared commmercially on a large scale as described by Brinkhous et al., J. Amer. Med. Assn. 205, 613-617 (1968) and in U.S. Pat. No. 3,631,018.
In order to obtain more highly purified concentrates of AHF, the cryoprecipitation procedures have been combined with fractionation methods that employ various chemical agents such as, e.g., ammonium sulfate, glycine, alcohol, heparin and polycondensed polyglycols such as polyethylene glycol (PEG), polypropylene glycol (PPG) and mixed polyethylene-polypropylene glycols, the latter being block copolymers of ethylene oxide and polyoxypropylene which are available commercially under the trademark Pluronic.RTM. from BASF Wyandotte Chemical Company, Further description of PEG and its use in the production of AHF from cryoprecipitate can be had by reference to U.S. Pat. No. 3,631,018, while similar disclosure of the Pluronic polymers and their use in the production of AHF from cryoprecipitate can be had by reference to U.S. Pat. No. 4,073,886. Disclosures of the use of heparin in the production of AHF from cryoprecipitate can be had by reference to U.S. Pat. Nos. 3,803,115, 4,203,891 and U.S. Pat. No. Re. 29,698.
It is known that during the production of AHF from cryoprecipitate there is an inevitable loss of AHF related to the further purification steps. Although these purification procedures may appear to be simple, they require great care to harvest an optimal amount of AHF. As noted recently by Johnson et al., of the New York University Medical Center, isolation of AHF by large scale techniques results in a maximum yield of about 30% and most large scale producers have obtained a yield of only 20% to 25%. Vox Sang. 36, 72-76 (1979). Thus it is desirable to develop some process which would allow removal of unwanted or unnecessary proteins (such as fibrinogen and its denatured and degraded products) from an AHF concentrate without undue loss of the valuable AHF itself. One approach to this goal is to produce a cryoprecipitate which contains increased amounts of AHF as described in recent U.S. Pat. Nos. 4,086,218; 4,105,650; 4,137,223; 4,189,425; and by Johnson et al., Vox Sang 36, 72-76 (1979). As disclosed in these patents and publication, small amounts of PEG and/or Pluronic polymers with or without added heparin are employed in the plasma prior to carrying out the freezing process to produce the cryoprecipitate. These procedures thereby result in a higher yield of cryoprecipitate and an associated greater amount of AHF. However, other proteins (e.g., fibrinogen and its derivatives) normally found in the cryoprecipitate are also proportionally increased. Methods normally used to remove the unwanted proteins (such as the precipitation with glycine, PEG and alcohol) unfortunately also remove a proportional amount of AHF by co-precipitation with no real net gain in the final yield of AHF.
Another blood protein which precipitates from plasma in the cold is known as cold-insoluble globulin (CIg or fibronectin). This is an opsonic plasma factor now identified as .alpha..sub.2 -surface binding globulin. Collection of the starting plasma in the aforesaid polycondensed polyglycols and/or heparin prior to cryoprecipitation also results in precipitation of the CIg with the AHF. Recent comments and reports on the importance of CIg have been published by Rock and Palmer, Thrombosis Res. 18, 551-556 (1980) and Anon., Hospital Pract. 4(7), 35-36 (1980). See also U.S. Pat. No. 4,210,580.